1. Field of the Invention
This invention relates to a method and compositions for determining the HLA DP genotype of an individual. In a preferred embodiment, the invention relates to using gene amplification methodology disclosed and claimed in U.S. Pat. Nos. 4,683,195 and 4,683,202 and the dot-blot and allele-specific oligonucleotide probe technology as disclosed and claimed in U.S. Pat. No. 4,683,194. The methods and probes of the invention specifically relate to the detection of the polymorphic class II HLA DP genes. The invention relates to the fields of molecular biology, diagnostic medicine, and forensics.
2. Description of the Related Art
The class II loci of the human major histocompatibility complex encode the HLA D cell surface glycoproteins which are expressed on B lymphocytes, activated T lymphocytes, macrophages, and dendritic cells. These proteins, which are individually designated DR, DQ, and DP, are composed of an alpha and a highly polymorphic beta subunit and are responsible for the presentation of antigen to T cells. The variability in the highly polymorphic beta subunit is localized to the amino-terminal extracellular domain, which is thought to interact with the T cell receptor and antigen peptide fragments. The genes encoding the class II HLA proteins are located on the short arm of chromosome six in humans. The genes encoding the HLA DPalpha and DPbeta chains are clustered at the centromeric end of this region and, perhaps due to low levels of expression, were the last of the HLA D genes to be discovered and are, therefore, the least well characterized. The structures, sequences, and polymorphisms in the HLA D region have been reviewed in Trowsdale et al., 1985, Immunol. Rev. 85:5-43, incorporated herein by reference.
The polymorphism of the HLA D region gene products has, in general, been defined by serologic typing reagents and by the mixed lymphocyte culture (MLC) reaction in which T cell proliferation in response to homozygous typing cells (HTC) is measured in culture. The HLA DP antigens were originally defined by their ability to stimulate a strong secondary response in specifically primed T cells, a method known as primed lymphocyte typing (PLT) and described by Mawas et al., 1981, Tissue Antigens 15:458-466; Wank et al., 1978, Immunogenetics 6:107-115;and Shaw et al., 1980, J. Exp. Med. 152:565-580. The HLA DP antigens elicit only a weak response in a primary MLC, and unlike the studies of HLA DR and HLA DQ polymorphism, the analysis of allelic variation in the HLA DP region has been complicated by the lack of availability of serologic reagents and of typing cells. In addition, the specific cell lines used in the PLT assay are difficult to generate, and the typing assay is slow and somewhat variable from laboratory to laboratory.
Cellular (see Odum et al., 1987, Tissue Antigens 29:101-109), biochemical (see Lotteau et al., 1987, Immunogenetics 25:403-407), and restriction fragment length polymorphism (RFLP, see Hyldig-Nielsen et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:1644-1648) analyses have indicated that the degree of polymorphism in the DP region may be more extensive than the currently serologically, immunologically, or PLT-defined DPw1 through DPw6 types. The RFLP approach showed that the RFLP of the DP region was relatively extensive and that some of the fragments were strongly associated with certain DP antigens. However, the RFLP technique has certain limitations. An allele carrying a variant sequence is identifiable only if the variant nucleotide is within the recognition site of a restriction enzyme used in the analysis or if a polymorphic restriction site is in linkage disequilibrium with a specific coding sequence variation. In addition, RFLP analysis simply provides evidence that a coding sequence variation exists but does not provide information on the exact nature of the variation. Moreover, relatively large fragments of the genomic nucleic acid must be used for the analysis. This latter requirement often rules out the use of samples which have been kept under conditions which result in the degradation of the genomic DNA.
Accurate DP typing may prove important in several medical applications. Genetic recombination between the DP loci and the serologically typed DR loci can occur such that serologically identical sibs may not be matched at DP. The HLA-DP differences have been revealed by RFLP analysis in several cases of acute graft vs. host disease between apparently HLA-identical donor and recipient pairs, as described by Amar et al., 1987, J. Immunology 138: 1947-1953. Furthermore, several autoimmune diseases, including coeliac disease, have been shown to be associated with specific DP types, defined either by PLT, Odum et al., 1986, Tissue Antigens 28:245-250, or by RFLP, Howell et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:222-226, analysis.
A. significant improvement in DNA amplification, the polymerase chain reaction (PCR) technique, was disclosed by Mullis in U.S. Pat. No. 4,683,202, and methods for utilizing PCR in the cloning and detection of nucleic acids were disclosed by Mullis et al. in U.S. Pat. No. 4,683,195, both of which patent disclosures are hereby incorporated herein by reference. See also U.S. Pat. No. 4,965,188 and abandoned U.S. Ser. No. 899,513, filed Aug. 22, 1986, each of which is incorporated herein by reference. In the PCR technique, short oligonucleotide primers are prepared to match opposite ends of the sequence to be amplified. The sequence between the primers need not be known. A sample of nucleic acid (DNA or RNA, although RNA is first convened to cDNA in the PCR process) is extracted and denatured, preferably by heat, and hybridized with oligonucleotide primers which are present in molar excess. Polymerization is catalyzed by a polymerase in the presence of deoxynucleoside triphosphates (dNTPs). This results in two "long products" which contain the respective primers at their 5'-termini, covalently linked to the newly synthesized complements of the original strands. The replicated DNA is again denatured, hybridized with oligonucleotide primers, returned to polymerizing conditions, and a second cycle of replication is initiated. The second cycle provides the two original strands, the two long products from cycle 1 and two long products of cycle 2, and two "short products" replicated from the long products produced in cycle 1. The short products contain sequences (sense or antisense) derived from the target sequence and flanked at the 5' end with a primer and at the 3'-end with a sequence complementary to a primer. On each additional cycle, the number of short products is replicated exponentially. Thus, the PCR process causes the amplification of a specific target sequence and allows for the detection of sequences initially present in a sample in only extremely small amounts.
Allelic sequence variations in the gene encoding beta-globin of hemoglobin and in the gene encoding HLA DQalpha have been detected by utilizing allele-specific oligonucleotides (ASO), which will only anneal to sequences that match them perfectly, as described by Saiki et al., 1986, Nature 324:163-166. These studies also utilized the PCR procedure to amplify the DNA sequences present in the samples and a dot-blot technique to detect probe hybridization to the sample.